Detection and quantification of nucleic acid molecules constitutes a fundamental element in several diagnostic techniques. An essential feature of such techniques is the ability of probes (a nucleic acid or nucleic acid analogue) to hybridize specifically to a complementary nucleic acid sequence. For hybridisation to occur some standard conditions have to be met regarding e.g. salt concentration and temperature, but the major determining factor is the number of fully matched nucleobases in the hybrid of two hybridizing strands. In hybrids of relatively short length, e.g. 6-10 basepairs, a single base pair mismatch will result in a drastic decrease in thermal stability, whereas the relative reduction of stability caused by a single base pair mismatch (or a deletion/insertion) becomes increasingly less with increasing length of the hybrid.
For diagnostic purposes, it is often desirable to identify a sequence of nucleobases which is present only in the gene or in the organism in question, but absent in any background nucleic acid that may be present in the sample. For any given sequence of nucleobases to be statistically unique in a typical sample, like the human genome, the length of the sequence will have to be in the order of 18-20 bases, which on the other hand will enhance its capacity to accomodate mismatches, without any major loss of thermal stability. Thus, smaller probes may have the disadvantage of not being statistically unique, whereas longer probes may have the disadvantage of not being able to discriminate mismatches because the overall stability of the hybrid is not significantly affected.